Hirudin is the most potent and specific inhibitor of the blood‐clotting enzyme thrombin so far known. Several hirudin variants were isolated mostly from Hirudo medicinalis and shown to be polypeptide chains of approximately 7 kDa with three internal disulfide bridges. In this study, limited proteolysis has been used to probe aspects of the structure and dynamics of a hirudin variant HM2 isolated from Hirudinaria manillensis. Proteolysis of the polypeptide chain of 64‐amino‐acid residues of hirudin HM2 by protease from Staphylococcus aureus V8, trypsin, thermolysin and subtilisin occurs at region 41–49 of the chain. The N‐terminal fragments 1–41 and 1–47 were isolated to homogeneity and shown to maintain inhibitory action on thrombin, though much lower than the intact protein. The results were interpreted on the basis of a proposed three‐dimensional structure of hirudin HM2 deduced by protein modelling the known structure of hirudin variant HV1 from Hirudo medicinalis (75% sequence similarity between HM2 and HV1). Both proteolysis experiments and protein modelling provide evidence for the existence in hirudin HM2 of a N‐terminal well‐structured domain (core) and a C‐terminal flexible polypeptide segment. Determination of the accessible surface area of the three‐dimensional model of hirudin HM2 showed that the sites of preferential cleavages are at the surface of the polypeptide molecule.
|Number of pages
|European Journal of Biochemistry
|Published - Dec 1994